This proposal concerns the artificially expanded genetic information system (AEGIS), a DNA-like molecular system having 8 letters in addition to the 4 in natural DNA. AEGIS displays five features of a synthetic genetic system (rule-based design, orthogonality, semi-predictable affinity, higher information density, and polymerase incorporability) that permits it to solve a range of problems in medical diagnostics, systems biology, and genomics. Today, over 400,000 patients have their health care improved due to AEGIS -enabled clinical assays that support personalized medicine. This project will move beyond AEGIS as a passive binding system to allow AEGIS components to participate as part of a dynamic system that exploits DNA polymerases and reverse transcriptases. This requires that we develop polymerases that incorporate AEGIS components with very high fidelity and efficiency. To obtain these, DNA polymerase that already do this reasonably well will be placed under selective pressure in a directed evolution experiment. The polymerases that are obtained will then be used in real time PCR instruments to develop nucleotide-polymerase-recipe combinations that support 6-letter polymerase chain reactions. Supporting technology, including the determination of fidelity of incorporation of these. We will then do a demonstration project to show that AEGIS components can solve a difficult problem in modern molecular, systems, and clinical biology: the multiplexing of PCR. Multiplexed PCR is the rate limiting step in many genomics analyses. Our long term, highly ambitious goal is to develop a "synthetic biology", a chemical system capable of Darwinian evolution, based on AEGIS components.